Project Summary Cardiac arrest (CA) has an incidence of 359,800 annually. Among survivors of CA, brain injury is the biggest impediment to functional recovery. Currently, neither pharmacological intervention nor therapeutic hypothermia can reverse the neural injury caused by CA. Stem cell therapy holds significant promise in the neuronal repair after brain injury. However, poor viability and integration at the site of injury and lack of efficient differentiation into the desired cell types hinder clinical applications. E merging metabolic glycoengineering (MGE) technology by modification of surface glycans impacts cell adhesion and differentiation in vitro, however, has not been investigated in the context of stem cell therapy. Therefore, the overall aim of this proposal is to apply MGE to cell-based therapies to improve cell adhesion and viability after transplantation and enhance the treatment efficacy to repair damaged neurons in ischemia brain after CA. The specific aims are: Aim1: With our novel MGE technique, we hypothesize that a novel glycan-based intervention is able to promote human neural stem cell (hNSCs) neural differentiation and cell adhesion in vitro. We will develop and optimize novel thiolated ManNAc analogs with longer alkyl chains, Ac5ManNPropT and Ac5ManNButT, that are predicted to increase thiol accessibility and promote hNSCs cell adhesion and neural differentiation in vitro. Aim2: With optimized ManNAc analogs, we hypothesize that treated hNSCs will promote the survival, distribution, and differentiation of transplanted hNSCs in vivo. We will evaluate the effect of glycoengineered hNSCs on functional outcome after CA and optimize this cell-based therapy. Aim 3: With expected improvement in outcome after CA, we hypothesize that the success of the cell- based intervention is due to improved survival and differentiation of transplanted glycoengineered hNSCs. We will explore cellular interactions and molecular mechanisms after glycoengineered hNSC transplantation after CA through Wnt/?-catenin signaling pathways. The Significance lies in the combination of the MGE technique and stem cell therapy for repairing brain injury post-CA, optimization of cell-based therapy towards clinical translation, and the expected discovery of the mechanism underlying improved survival and differentiation after glycoengineered NSC transplantation. The innovation lies in our innovative hypothesis to modify stem cell surface properties by MGE technology to improve cell survival and differentiation, our novel and effective MGE method with low cost for modifying surface glycans of hNSCs, and our use of the MGE technique in important disease in vivo model to develop novel therapeutic cell-based intervention. Our study will lead to the development of novel therapeutic strategies to repair brain injury towards future clinical interventions and maximize the benefits of MGE and stem cell therapy based on the new findings. The use of sugar analog molecules for regenerative medicine and stem cell therapies will help improve cells based therapy to repair brain injury due to CA, stroke, and trauma, or neurodegenerative diseases, and have tremendous potential to provide a profound medical advance.